The present invention relates to transgenic zoysiagrass (Zoysia japonica Steud.) comprising plant cells transformed with a recombinant DNA construct containing a nucleotide sequence which encodes a modified oat phytochrome A, in which serine-598 was substituted with alanine to improve light responsiveness. The resultant transgenic zoysiagrass exhibits significant reduction of typical shade avoidance reactions, such as dwarfish internodes and petioles, short leaves, dark-green leaf color, and healthy and robust appearance, which greatly affects yields and environmental adaptation of crop plants.
Zoysiagrass, which is also known as Korean grass, is one of the most widely cultivated turfgrass species in the temperate zone worldwide, especially in the Far-Eastern Asia, including Korea, Japan, and Eastern area of China. The cultivation area is rapidly expanding in recent years in USA and other countries due to its unusual characteristics such as drought resistance and rapid recovering capacity from traffic damages. Particularly, it grows well even in poor soil under virtually all climates. It is therefore widely used for both practical and decoration purposes, such as in golf courses, athletic fields, roadsides, home gardens, and riverbanks. However, zoysiagrass has some intrinsic drawbacks whose resolution is strongly demanded by the customers. Major concerns include improved resistance to pathogens and herbicides and adaptive plasticity to environmental changes. In addition, zoysiagrass tends to overgrow during the hot summer season and require frequent watering and mowing that is labor-consuming and costly. Of particular concern is that the overgrown plant is very susceptible to fungal infections, and vast amount of chemicals, including fungicides, should be spread to control various pathogens, which resultantly causes a serious environmental pollution. One efficient way to solve these problems in zoysiagrass cultivation is to genetically engineer it so that it exhibits reduced shade avoidance responses. Shade avoidance is an adaptive mechanism for plants to overcome severe light competition occurring in nature (Smith and Whitelam, 1997). When plants grow in the dark or shade, which is naturally represented under the canopy of neighboring plants, stem or hypocotyl extension is drastically stimulated at the expense of leaf and root growth, resulting in elongated but weak and pale appearance. As a result, they are very susceptible to pathogen infections and environmental stress, causing significant loss of crop productivities. Genetically engineered zoysiagrass with reduced shade avoidance reactions will grow slower but healthier than parental plants. With well-established plant molecular biological and plant tissue culture techniques available to plant biologists, it is now possible to introduce any particular gene of interest into virtually any crop plant and to introduce or improve useful traits in a predictable way.
Plant growth and development is regulated through coordinate interactions between intrinsic developmental cues, such as growth hormones, and various environmental factors. Light is one of the most important environmental factors that influences many aspects of plant growth and developmental processes throughout the whole life span, covering from seed germination, leaf and stem growth, photoperiodism, shade avoidance, to flowering, which are collectively called photomorphogenesis. One prominent photomorphogenic response critical for survival and propagation for plants in nature is the shade avoidance. It is a well-known plant vegetation dynamic process that is absolutely required to overcome severe light competition occurring in nature, especially when plants grow in close proximity, as naturally occurring in forests and frequently encountered in densely cultivated area. Shade avoidance response is typified by abnormally fast extension growth of stems and petioles but with suppressed leaf and root growth and storage organ production to get sufficient light required for photosynthesis, resulting in elongated but weak and pale appearance. Although it is an essential mechanism for plant survival, it could be a potential problem in crop cultivation, since crop plants become slender, weak, and pale and susceptible to pathogen infections and environmental changes, such as rain and wind. There have been diverse efforts to resolve this problem in crop cultivation, and it has been recently found that manipulation of shade avoidance reactions is a promising way to achieve this.
The proximity signal that induces shade avoidance reactions in plants is the radiation reflected from neighboring plants. Photosynthetic pigments absorb most of the visible light wavelengths (400–700 nm), but lights with longer wavelengths (700–800 nm) in the far-red light range is reflected or transmitted, resulting in prevalence of far-red (FR) light wavelength under canopy. FR light is perceived by the phytochrome photoreceptors. The physiological function of the phytochromes is primarily driven by a unique photochemical activity, reversible phototransformation between two distinct spectral forms, the red (R) light absorbing Pr form and the FR light absorbing Pfr form. The Pfr form is responsible for physiological functions in most photomorphogenic processes. It is therefore evident that the phytochrome action is determined by the R:FR ratio in the light environment. There are at least five different phytochromes (phyA to phyE) known in Arabidopsis. They exert both overlapping as well as distinct functions. In etiolated plants, phyA are accumulated to a high level and inhibits stem growth in response to light, with the λmax being around 710–720 nm. However, in light-grown plants, phyA is rapidly degraded, and phyB has a primary role in modulation of stem growth as a function of R:FR ratio. When FR content is high, as occurred under canopy, phyB modulates the shade avoidance reactions. As a consequence, FR light inhibits stem growth mainly via phyA action in etiolated plants but stimulates stem growth in light-grown plants primarily via phyB action.
It has been clearly established that in transgenic plants overexpressing phyA, the phyA level is maintained to a certain level sufficient to offset light-induced degradation and exhibits a severe dwarfism when grown in light environment that contains relatively high content of FR wavelength. Although engineering of shade avoidance by introducing a foreign PHYA gene was successful in dicotyledonous plants (Boylan and Quail, 1989), transgenic rice plants that overexpress a PHYA gene did not show such a dwarfish appearance. Two possibilities have been suggested to explain the absence of dwarfish appearance in transgenic rice plants. First, monocotyledonous or dicotyledonous phytochrome may have a distinct function, each specifically functioning in each plant group. Alternatively, the expression level of the phyA was not enough to offset the light-induced degradation of the intrinsic phyA, possibly due to inefficient action of the promoter employed. Since all phyAs isolated from both monocotyledonous and dicotyledonous plants show high sequence homologies and exhibit identical photochemical activities, we assumed that the latter might be the reason for the absence of any phenotypic alterations in the transgenic rice plants expressing a foreign phyA.
We recently obtained an oat phyA mutant, in which serine-598 was substituted with alanine, that showed a hypersensitive light response. Transgenic model plants expressing the modified phyA were more severely dwarfish than those expressing a wild type phyA when grown in the light. Based on this observation, it was anticipated that transgenic turfgrass or rice with the modified phyA would cause reduced shade avoidance reactions, more prominently than that with the wild type phyA, irrespective of the presence or absence of neighboring plants.
As used herein, the term “genetic transformation” refers to a procedure to introduce a gene(s) or genetic material(s) into a crop plant of interest in a predictable way. The gene or genetic material is stably integrated into the plant genome and transmitted through generations as a part of the plant genome.